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DOI: 10.4172/pharmaceutical-sciences.1000393

Abstract

Saposhnikovia divaricata, Peucedanum japonicum, and Glehnia littoralis of the family Umbelliferae, have long been used as traditional herbal medicine in Asian countries. This study is the first to compare and analyze the relationship between antimicrobial activity, antioxidant activity, and total polyphenol content of Saposhnikovia divaricata, Peucedanum japonicum, and Glehnia littoralis. The four fractions of hydro-methanol extract of these 3 plants were evaluated using the disc diffusion method to determine the minimum inhibitory concentration against 5 different bacterial strains. The ethyl acetate fraction was the most effective against the bacterial strains investigated. The 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging activity was evaluated to measure antioxidant activity. The antioxidant activity of the ether and ethyl acetate fractions was in the order of Saposhnikovia divaricata>Peucedanum japonicum>Glehnia littoralis. The total polyphenol content of Saposhnikovia divaricata was greater than that of Peucedanum japonicum and Glehnia littoralis. Even though the 3 plants belong to the same family and are used for similar medical purposes, their antimicrobial activity, antioxidant activity, and total polyphenol content was different. This study would help researchers to uncover the critical aspects of the activities possessed by plants.

Keywords

Saposhnikovia divaricata (also known as Ledebouriella
seseloides, SD), Peucedanum japonicum (PJ), and
Glehnia littoralis (GL) have long been used in
traditional medicine under the name of Bangpung in
Korea, which is also known as Fang Feng in traditional
Chinese herbal medicine and Bofu in Japan. These
plants belong to the Umbelliferae family and are
assumed to have similar effects. These are commonly
used as ingredients in many polyherbal preparations
designed to dispel “wind’’ and to induce sweat, alleviate
rheumatic conditions, and relieve spasms. They have
also been used to treat cough and neurological diseases.
The active constituents reported from these plants were
essential oils, mannitol, bitter glycoside, chromones,
coumarins, and polyacetylenes [1].

Investigations of the antimicrobial activity of medicinal
plants has been an ongoing activity. Compounds isolated
from plants that possessed antimicrobial activity include
terpenoids, saponins, phenolics and phenylpropanoids.
Many plants used as aromatic herbs and spices have
been reported to have antimicrobial activity and
antioxidant activity, while phenolic compounds are
often used as aromatizants or antioxidants in the food
industry [2-6]. It is well-known that free radicals induce
oxidative stress, which might cause damage to lipids,
proteins, and nucleic acids in the body and result in
various diseases. The harmful effects of free radicals
can be attenuated by antioxidants, and the 1,1-diphenyl-
2-picryl-hydrazyl (DPPH) free radical scavenging
assay is a common method to determine the antioxidant
potential of plant extracts [7-9]. Antioxidant activity of
phenolic compounds widely distributed in plants could
be of value to human health. Total phenolic content
and antioxidant activities are strongly correlated, and a relationship between phenolic compounds and
antimicrobial activity has also been reported [10-13].
However, no studies have compared the antimicrobial
activity, antioxidant activity and phenolic contents of
SD, PJ and GL.

The main components of SD, PJ and GL reported
were phenolics including coumarine derivatives
(psoralen, bergapten, imperatorin), coumarin
isomer, chromone and its derivatives (divaricatol,
ledebouriellol, hamaudol) and flavonoids (rutin,
ferulate). These phenolic compounds are known to
have various biological effects including analgesic and
antiinflammatory activity [14,15].

The present study was conducted to assess the
differences in the antimicrobial activity, DPPH free
radical scavenging activity, and total polyphenol
content of the three aforementioned species of the
Umbelliferae family. To accomplish this, the disc
diffusion method was applied to measure the minimum
inhibitory concentration (MIC) of partitioned fractions
of the methanol extract against five different bacterial
strains, including three Gram-positive and two Gramnegative
bacteria. To measure the radical scavenging
activity, we used a DPPH assay, while the total
polyphenol content was determined using the modified
Folin-Denis method [16]. L-Ascorbic acid, DPPH, Folin-
Denis’ reagent, tannic acid and sodium carbonate were purchased from Sigma-Aldrich. All solvents and other
chemicals were purchased from Dae-Jung and Junsei
Chemical.

SD root was purchased from a commercial market,
while PJ and GL were collected from Jindo Island
in the southern portion of Korea. The materials
were authenticated by Prof. K. W. Yun and Voucher
specimens were deposited in the Herbarium of Sunchon
National University, Korea. The root part of the plants
was used for all experiments. The collected samples
were air-dried for 14 d, after which they were extracted
with hydro-methanol.

The air-dried roots were pulverized using an electric
mill, after which 200 g of the powdered root was
macerated with 1000 ml of methanol/water (80:
20 v/v) for 24 h. The percolates were then filtered
through Whatman No. 2 filter paper. Next, the crude
hydro-methanol extract was subsequently fractionated
with 500 ml of hexane, after which the top hexane layer
(comprising the hexane fraction) was concentrated.
The remaining layer was successively partitioned
with 500 ml of diethyl ether, ethyl acetate and water
in a separating funnel (forming the ether, ethyl acetate
and water fractions). Each fraction was subsequently
concentrated in vacuo to 30 ml at 30° and tested
for antimicrobial, antioxidant activity and the total
polyphenol content.

Each bacterial strain was grown in a nutrient broth at
30° for 18-24 h prior to testing, then subcultured three
times for another 18-24 h. The turbidity of bacterial
cell suspensions was brought to 0.3 optimal density at
660 nm by adding sterile broth and was then used for
the tests. Next, 10 ml of the bacterial cell suspensions
was poured uniformly onto nutrient agar plates and
then paper disks (8.0 mm in diameter) containing the
hexane, ether, ethyl acetate, or water fractions of the
extracts were carefully placed on the bacteria-seeded
Petri dishes. The diameters of the resulting zones of
inhibition were measured in mm after the cultures
were incubated for 24 or 48 h at 30°. The diameter
of inhibition zone was measured [17,18]. The MIC was determined as the lowest concentration that caused an
inhibition zone.

The DPPH free radical scavenging activity was
evaluated using the Blois method (1958) [19], with slight
modification. Briefly, 160 μl of each fraction sample
was mixed with 40 μl of 1.5×10-4 M DPPH solution (in
methanol) solution. The mixtures were gently mixed
and allowed to stand at room temperature for 30 min,
and the absorbance at 520 nm were measured using
a microplate spectrophotometer reader (Molecular
Devices). The antioxidant activity of each fraction was
expressed in terms of IC50 values (the concentration
required to inhibit DPPH radical formation by 50 %).
L-Ascorbic acid was used as reference.

The total polyphenol content was determined using the
Folin-Denis method, with slight modification [16]. The
fractions were centrifuged at 1200 rpm for 10 min,
and the supernatant collected. The clear supernatant of
the samples (0.5 ml) were mixed with 8 ml of distilled
water, after which 0.5 ml of Folin-Denis’ reagent
was added. After 3 min, 1 ml of sodium carbonate
(10 % in distilled water) was added and the solution
was allowed to stand for 2 h at 22° in darkness. The
absorbance was then measured at 700 nm using an UVVis
spectrophotometer (HP-8453, USA). A standard
curve prepared from tannic acid (50-300 mg/l) was
used for quantification and the total polyphenol content
was expressed as mg tannin/g dry weight.

All tests and analyses were carried out in triplicate and
each experiment was repeated three or four times. The
data shown here represent the mean ± standard deviation.
The statistical significance was determined by analysis
of variance (ANOVA) followed by Duncan’s multiple
range test.

The antimicrobial activity and MIC of fractions of
methanol extracts from the three test plants root are
shown in Table 1. The ether and ethyl acetate fractions
of SD and PJ showed activity, while GL primarily
showed activity in the ethyl acetate fraction. Overall, the
ethyl acetate fraction showed a strong inhibitory effect
against bacterial growth and Gram-negative bacteria
were more sensitive than Gram-positive bacteria to
the three test plants. The MIC values indicated that E. coli was the most sensitive investigated
microorganism (MIC=0.1 mg/ml) to all three test
plants. Additionally, SD and PJ were more effective
against both Gram-positive and Gram-negative
bacteria, while GL was only effective against Gramnegative
bacteria. In the case of PJ, the ethyl acetate fraction was effective against all five bacterial strains
at a MIC of 0.1 mg/ml (Table 1).

Species

Minimum Inhibitory Concentration (MIC, mg/ml)

Gram-positive

Gram-negative

B. subtilis

S. aureus

L. monocytogene

S. typhimurium

E. coli

S. divaricata

Hexane

1.5

-

-

-

0.2

Ether

0.5

0.3

2.0

0.2

0.2

Ethyl acetate

0.2

0.2

0.2

0.1

0.1

Water

1.0

-

-

-

-

P. japonicum

Hexane

-

-

-

-

0.3

Ether

2.0

0.5

1.5

0.5

0.3

Ethyl acetate

0.1

0.1

0.1

0.1

0.1

Water

-

-

-

-

-

G. littoralis

Hexane

-

-

-

-

-

Ether

-

-

-

-

0.5

Ethyl acetate

2.0

1.5

-

0.2

0.1

Water

-

-

-

-

-

‘-’ not detected

Table 1: MIC of each fraction of methanol extracts of S. Divaricata, P. Japonicum and G. Littoralis against gram-positive and gram-negative bacteria

Antioxidant activity was determined by DPPH assay
and expressed as IC50 (μg/ml), which was the amount of
sample needed to scavenge 50 % of initial concentration
of the free radical. Results of the IC50 values revealed
that ether and ethyl acetate fraction of methanol
extract from the three plants had higher scavenging
activity when compared with the hexane and water
fraction (Table 2). Specially, the order of DPPH
antioxidant activity of the ethyl acetate fraction was PJ
(5.42 μg/ml)>GL (16.66 μg/ml)>SD (30.54 μg/ml; Table 2).

Species

IC50 (μ/ml)a

Hexane

Ether

Ethyl acetate

Water

Ascorbic acid

S. divaricata

145.53 ± 12.17

5.30 ± 0.05

30.54 ± 1.33a

186.91 ± 4.28a

3.04 ± 0.05

P. japonicum

246.71 ± 43.65a

46.03 ± 5.85

5.42 ± 0.05

104.97 ± 5.90

G. littoralis

44.64 ± 3.22

71.43 ± 16.00a

16.66 ± 0.29

-

aMeans ± standard deviation with the same letters within a column are not significantly different at p=0.05 according to Duncan’s multiple range test. ‘-’ not detected

Phenolic compounds have been investigated in many
medicinal plants. The total phenolic content of the
three plants was shown in Table 3. It was observed that
the amount of total phenolic content in ether and ethyl
acetate fraction was high, and low in hexane and water
fraction. It showed that in ethyl acetate fraction of PJ
(57.42 mg/g) presented higher total phenolic followed
by GL (31.66 mg/g) and SD (26.32 mg/g).

Species

Total polyphenol content (mg/g dw)a,b

Hexane

Ether

Ethyl acetate

Water

S. divaricata

12.53 ± 2.17a

53.30 ± 1.24a

26.32 ± 0.91b

7.81 ± 2.78b

P. japonicum

6.71 ± 2.65b

21.71 ± 2.16b

57.42 ± 2.05a

12.97 ± 1.41a

G. littoralis

17.64 ± 1.22a

11.43 ± 1.57

31.66 ± 1.31b

2.42 ± 0.81

aMeans ± standard deviation with the same letters within a column are not significantly different at p=0.05 according to Duncan’s multiple range test. bdw : dried weight

Table 3: Total Polyphenol content of each fraction of methanol extract of S. divaricata, P. japonicum and G. littoralis

Plant derived bioactive substances especially with
antioxidant and antimicrobial activities could be of
potential use to pharmaceutical and food industry
and to the consumers [4,20,21]. SD, PJ and GL have
been used in a variety of herbal medicines under
the assumption that they all have similar effects.
However, the present study demonstrated that there
are notable differences among the antimicrobial
activities, DPPH free radical scavenging activities
and total polyphenol contents of these plants. SD
is known to have tumor cell growth inhibitory
effects and antioxidant activities. Additionally,
chromone extract of SD has antirheumatoid effects
that occur via inhibition of nuclear factor-kappaB
(NF-kB) and mitogen-activated protein kinases
(MAPK) [9,22,23]. PJ has antiobesity effects that occur
via phenolic compounds such as neochlorogenic acid,
chlorogenic acid and rutin, as well as antiplatelet
activity induced by khellactone and monoamine oxidase
inhibitory effects exerted by coumarine [24-27]. GL has
antioxidant effects, antiinflammatory effects that occur
via coumarin derivative imperatorin, antibacterial and antifungal effects, and antiinflammatory activity
owing to NF-kB and MAPK activity. GL has polyine
compounds, which are not phenolic, but have
been reported to have antibacterial and antifungal
activities [28-32]. Other active constituents of GL include
lignin and neolignan (polyphenolic substance) and
biphenyl ferulate (flavonoid) [14,15]. The antimicrobial
effects of the three plants could be of use against
infectious disease. In the present study, the disc
diffusion method indicated that ethyl acetate fraction is
the most effective fraction against both Gram-positive
and Gram-negative bacteria. Overall, Gram-negative
bacteria were more sensitive to the three tested plants
than Gram-positive bacteria, since the fractions of
the three plants exhibited lower MIC values against
Gram-negative bacteria. However, for Gram-positive
bacteria, SD and PJ showed better effect than GL.
Due to the differences in the activity and chemical
properties, these plants should be used for specific
purposes when employed as food additives and/or for
medicinal purposes. DPPH free radical scavenging
activity measures the degree of inhibition of DPPH
free radicals. Regarding the antioxidant activity, SD in
ether fraction and PJ in ethyl acetate fraction exhibited
the highest antioxidant activity. The relation between
antioxidant and total phenolic content for the three
plant examined was shown (Table 2 and 3). All the
three plants have antioxidant activity and their active
components might be related to phenolic compounds,
which have antioxidative and antimicrobial activity [13].
Lipopolysacchride (LPS) is a component of cell
wall of Gram-negative bacteria and uses commonly
for inflammation-associated researches. Anomalin,
isolated from SD and a pyranocoumarin derivative,
has antiinflammatory effect against LPS-induced
macrophage activation. Also there is a report of
antiinflammatory effect by GL on LPS-treated RAW
264.7 cells. Interestingly, there are no reports on the
LPS or inflammation related research from SD [29,32].

The results of the present study suggest that there are
differences in the antimicrobial activity, antioxidant
activity, and total polyphenol content among the three species of plants in the Umbelliferae family, which
should be considered prior to their use for specific
biotechnological, nutraceutical or pharmaceutical
applications. It should be noted that this is a preliminary
study and further investigations are needed to determine
the mechanism of action of the investigated plants.

Conflicts of interest

The authors declare that there are no conflicts of
interest.

Financial support and sponsorship

This study was supported by the National Research
Foundation of Korea Grant funded by the Korean
Government (NRF-2007-361-AM0015) and Suncheon
Research Center for Natural Medicines.

About the Journal

The Indian Journal of Pharmacy was started in 1939 as "a quarterly journal devoted to the Science and practice of Pharmacy in all its branches". The Chief editor and the main guiding force behind the 'Journal' was Prof. M.L. Schroff, Head of the Department of Pharmaceutics. Benaras Hindu University, Benaras. more